Apoptosis, also referred to as physiological cell death or programmed cell death, is a normal physiological process of cell death that plays a critical role in the regulation of tissue homeostasis by ensuring that the rate of new cell accumulation produced by cell division is offset by a commensurate rate of cell loss due to death. Apoptosis can be characterized by morphological changes in the cell, including fragmentation of nuclear chromatin, compaction of cytoplasmic organelles, dilatation of the endoplasmic reticulum, a decrease in cell volume and alterations to the plasma membrane, resulting in the recognition and phagocytosis of apoptotic cells and prevention of an inflammatory response. Disturbances in apoptosis that prevent or delay normal cell turnover can be just as important to the pathogenesis of diseases as are known abnormalities in the regulation of proliferation and the cell cycle. Like cell division, which is controlled through complex interactions between cell cycle regulatory proteins, apoptosis is similarly regulated under normal circumstances by the interaction of gene products that either induce or inhibit cell death.
The stimuli that regulate the function of these apoptotic gene products include both extracellular and intracellular signals. Either the presence or the removal of a particular stimulus can be sufficient to evoke a positive or negative apoptotic signal. Physiological stimuli that inhibit or reduce the likelihood of apoptosis include, for example, growth factors, extracellular matrix, CD40 ligand, viral gene products, neutral amino acids, zinc, estrogen and androgens. In contrast, stimuli that promote apoptosis include, for example, tumor necrosis factor (TNF), Fas, transforming growth factor xcex2 (TGFxcex2), neurotransmitters, growth factor withdrawal, loss of extracellular matrix attachment, intracellular calcium and glucocorticoids. Other stimuli, including those of environmental and pathogenic origin, also exist and can either induce or inhibit apoptosis. Although apoptosis is mediated by diverse signals and complex interactions of cellular gene products, the results of these interactions ultimately lead into a cell death pathway that is evolutionarily conserved between humans and invertebrates.
Several gene products that modulate the apoptotic process have been identified. Although these products can, in general, be separated into two basic categories, gene products from each category can function to either inhibit or induce apoptosis. One family of gene products is the Bcl-2 family of proteins. Bcl-2 is the best characterized member of this family and inhibits apoptosis when overexpressed in cells. Other members of the Bcl-2 family of proteins include, for example, Bax, Bak, Bcl-xL, Bcl-xS and Bad. While some of these proteins can inhibit apoptosis, others can induce apoptosis (for example, Bcl-xS and Bak, respectively).
A second family of gene products, the caspase family, is related genetically to the C. elegans ced-3 gene product, which is required for apoptosis in the roundworm, C. elegans. The caspase family includes, for example, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9 and caspase-10. Among the common features of the caspase gene products is that 1) they are cysteine proteases with specificity for substrate cleavage at Asp-X bonds, where xe2x80x9cXxe2x80x9d is an amino acid; 2) they share a conserved pentapeptide sequence within the active site; and 3) they are synthesized as proenzymes that require proteolytic cleavage at specific aspartate residues for activation of protease activity. Cleavage of the proenzyme produces two polypeptide protease subunits, which combine non-covalently to form a tetramer comprised of two heterodimers. Although these proteases, when expressed in cells, induce apoptosis, several alternative structural forms of these proteases, such as caspase-1xcex4 (ICExcex4), caspase-1xcex5 (ICExcex5), caspase-2S (ICH-1S), caspase-6xcex2 (Mch2xcex2) and caspase-7xcex2 (Mch3xcex2), inhibit apoptosis.
In addition to the Bcl-2 and caspase families, which play a role in apoptosis in mammalian cells, other gene products are important in mammalian apoptosis. For example, in addition to ced-3, another C. elegans gene product, ced-4, is required for apoptosis in C. elegans. Apaf-1, a human protein homologous to ced-4, binds cytochrome c and may activate caspase-3, leading to apoptosis. In addition, another protein, casper, while not a caspase, has sequence similarity to caspase-8 throughout its length and interacts with caspase-8 and caspase-3 through distinct domains. Overexpression of casper in mammalian cells induces apoptosis.
It is uncertain whether other genes encode members of either of the Bcl-2 or caspase gene families and, if so, what role they play in the apoptotic pathway. It also is unclear what physiological control mechanisms regulate apoptosis and how the apoptotic pathways interact with other physiological processes. For example, it has been suggested that cytotoxic T lymphocytes mediate their destructive function by inducing apoptosis in their target cells.
The process of apoptosis maintains tissue homeostasis in various physiological processes, including embryonic development, immune cell regulation and normal cell turnover. It follows that the loss of apoptosis can lead to a variety of pathological disease states. For example, the inappropriate loss of apoptosis can lead to the pathological accumulation of self-reactive lymphocytes such as those occurring in association with many autoimmune diseases. Inappropriate loss of apoptosis also can lead to the accumulation of virally infected cells and of hyperproliferative cells such as tumor cells. Similarly, the inappropriate activation of apoptosis can contribute to a variety of pathological disease states including, for example, acquired immunodeficiency syndrome (AIDS), neurodegenerative diseases and ischemic injury. Treatments that are specifically designed to modulate the apoptotic pathways in these and other pathological conditions can change the natural progression of many of these diseases.
Thus, there exists a need to identify apoptotic genes and their gene products and for methods of modulating apoptosis for the therapeutic treatment of human diseases. The present invention satisfies this need and provides related advantages as well.
The invention generally provides caspase-14. In one aspect, the invention provides an isolated nucleic acid molecule encoding a caspase-14 polypeptide or a functional fragment thereof. Nucleic acid and amino acid sequences of caspase-14 are provided. The invention also provides caspase-14 polypeptides or a functional fragment thereof.
In another aspect, a vector that contains the nucleic acid molecule and a host cell that contains the vector is also provided. Also provided is an expression vector comprising the nucleic acid molecule encoding caspase-14 that is operatively linked to a promoter.
In other aspects, an isolated caspase-14 polypeptide and functional fragment thereof are also provided, as are antibodies that specifically bind thereto. In addition, the invention provides methods of identifying compounds that modulate caspase-14 activity comprising: (a) contacting a sample containing a caspase-14 polypeptide or functional fragment thereof with a test compound, and thereafter (b) determining the activity of caspase-14 polypeptide or functional fragment thereof.
Methods are also provided for identifying inhibitors and enhancers of caspase-14 activity, comprising: (a) contacting an activated caspase-14 polypeptide with a substrate in the presence of a test compound under conditions in which the caspase-14 processes the substrate in the absence of the test compound; and thereafter (b) detecting increased or decreased substrate turnover, wherein increased substrate turnover indicates the presence of an enhancer and wherein decreased substrate turnover indicates the presence of an inhibitor.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, the various references set forth below that describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated herein, by reference, in their entirety.